We previously reported that Chlamydia trachomatis hypothetical protein CT311 was secreted out of chlamydial inclusion and into host cell cytosol. We now found that CT311 further entered host cell nucleus at the late stage of infection and continued to accumulate in the nucleus of C. trachomatis-infected cells. When CT311 was expressed via a transgene in mammalian cells, CT311 protein was exclusively detected in the nucleus, suggesting that CT311 by itself is sufficient for nuclear targeting. However, preexisting nuclear CT311 did not affect subsequent chlamydial infection. Using deletion constructs, we mapped a nuclear localization signal sequence of CT311 to residues 21 to 63 (21AVEGKPLSRAAQLRERRKDLHVSGKPSPRYALKKRALEAK？KNK63). This sequence was sufficient for targeting a heterologous protein into mammalian cell nucleus and it contains two independent clusters of basic residues (34RERRK38 and 53KKRALEAKKNK63 respectively). Deletion or alanine substitution of the basic residues in either cluster led to loss of nuclear targeting activity, suggesting that both clusters are critical for the nuclear targeting function. These observations have demonstrated that the hypothetical protein CT311 possesses a novel nuclear localization signal sequence with dual modules of basic residues for targeting host cell nucleus during Chlamydia trachomatis infection.
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